Project Summary This project aims to understand how coordinated regulation of two key signaling pathways regulates differentiation and tumor grade in salivary gland mucoepidermoid carcinoma (MEC). Alterations in the activation of these pathways produce the extreme intra-tumoral cellular heterogeneity characteristic of salivary MEC tumors. These tumors are marked by the presence of variable proportions of three major cell types: an epidermoid or squamous cell type, a mucous-containing cell type, and an intermediate cell type. Intermediate cells represent a poorly differentiated cell type that can give rise to both epidermoid- and mucous-type cells. In more aggressive high-grade MECs, the intermediate cell tends to predominate, while mucous and epidermoid cells are more prevalent in low-grade MECs, highlighting the need to understand the drivers regulating differentiation in these tumors. Notably, over 50% of salivary MECs are characterized by a t(11;19) chromosomal translocation that results in a CRTC1/MAML2 (C1/M2) fusion oncoprotein. Recent whole-exome sequencing data from human MEC salivary tumors has shown that copy number variations and somatic mutations are lacking in most C1/M2-positive tumors, suggesting that C1/M2 is the main oncogenic driver in these salivary MECs. Previous research has demonstrated that C1/M2 triggers CREB-dependent gene expression and also interacts with MYC, amplifying expression of MYC transcriptional targets. Both CREB and MYC play key roles in controlling differentiated or stem-like states, respectively, in epithelial cells. Thus, the degree to which C1/M2 regulates targets within each pathway may influence the differentiation state of tumor cells, enabling the rise of a heterogeneous population of cell types within a single tumor. Mechanistically, the differential activation of CREB or MYC target genes by C1/M2 may regulate the progression of these cancers from low to high-grade, therefore, the working hypothesis for this proposal is that variations in CREB and MYC regulation accounts for the cellular heterogeneity observed in MEC tumors. To test these hypotheses, this proposal aims to (1) isolate and phenotypically characterize the epidermoid, mucous, and intermediate cell types and (2) explore the relative contributions of CREB- and MYC-driven pathways to the different cell types observed in MEC tumors.